Nef is one of four accessory proteins that enable HIV-1 to undermine host defenses. Nef is not targeted by any current antivirals, but is an exciting potential target because it is essential for the infectivity of virions. Long term infection with HIV strains bearing defective nef alleles progresses to AIDS very slowly, if at all. Many functions have been imputed to Nef, including modulating signaling by protein tyrosine kinases and downregulating CD4, MHC-I, BST2/tetherin (for O-group Nefs) and other cell surface receptors. On the basis of breakthrough findings in 2015, the mechanism whereby Nef confers infectivity on HIV-1 was shown to be the downregulation of the integral membrane protein, SERINC5. SERINC5 contains ten putative transmembrane spanning helices and no motifs suggestive of a binding site for Nef. Little biochemical literature exists on SERINC5 and it is currently unclear whether SERINC binds Nef directly or via one or more additional factors. Of the additional factors involved in Nef downregulation of SERINC5, the tetrameric clathrin adaptor AP-2 is known to be essential for SERINC5 downregulation and to directly bind to Nef. AP-2 is involved in clathrin-mediated endocytosis, but is not directly involved in degradative sorting. Another factor, Alix, is associated with the ESCRT machinery of lysosomal sorting, and interacts with Nef. Nef interacts with Alix in the context of the Gag-Pol fusion. All of these proteins function in the context of cell membranes. Aim 1 of this project will reconstitute the interaction network of SERINC5, AP-2, Alix, and Gag-Pol. Nef downregulates MHC-I via the clathrin adaptor AP-1, a process that is critically dependent on the small G-protein Arf1. Arf1 does not promote AP-2 dependent endocytosis, however. In Aim 2, we will isolate clathrin-coated vesicles formed by AP-2 upon Nef induction and compare their components to AP-2/clathrin vesicles in the absence of Nef. This will allow us to identify the putative Arf1 counterpart for Nef-dependent AP-2 vesicle formation. The novel component(s) will then be incorporated into the reconstitution experiments in Aim 1, bringing the project full circle. In Aim 3, we will refine conditions for expression of several SERINCs including 3,5 seeking a pure homogeneous and stable product and complex formation for structure analysis.